More than 60 million people worldwide have been infected by the human immunodeficiency virus (HIV) and nearly half have died of the resultant Acquired Immunodeficiency Syndrome (AIDS) since 1981. About 2.7 million new infections were reported in 2007. Therefore, development of an effective and safe HIV vaccine is urgently needed to contain the spread of HIV and AIDS. However, most previous efforts to achieve this goal have failed. Clinical trials of the first T-cell vaccine (Merck's MRKAd5 HIV-1 gag/pol/nef trivalent vaccine) were terminated recently because the data showed that the vaccine was unable to prevent HIV infection and could not lower virus levels in vaccinated volunteers who became infected. The first B-cell vaccine (VaxGen's AIDSVax, a bivalent gp120-based subunit vaccine) tested in clinical trials also failed to protect volunteers from HIV infection, possibly due to its inability to elicit broad neutralizing antibody responses because of great variability and high glycosylation of gp120. Since the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 has relatively conserved sequence and less glycosylation sites than gp120, it may be a better target than gp120 for vaccine development. Indeed, two human monoclonal antibodies (mAbs) targeting gp41, 2F5 and 4E10, exhibit much broader neutralizing activity than those targeting gp120 (mAbs 2G12 and b12).
HIV-1 gp41 (SEQ ID NO. 2) plays an essential role in virus fusion with the target cell. HIV-1 pg41 consists of three essential functional regions: fusion peptide (FP), N-terminal heptad repeat (called NHR or HR1, which refer to the same sequence and are considered equivalent as used herein) and C-terminal heptad repeat (called CHR or HR2, which refer to the same sequence and are considered equivalent as used herein) (FIG. 1A). Both NHR and CHR contain a number of leucine zipper-like motifs which have tendency to form coiled coil structures. Peptides derived from the NHR and CHR regions are effective in inhibiting HIV-1 fusion with the target cells and one of the CHR-peptides, enfuvirtide was licensed by the United States Food and Drug Administration (US FDA) in 2003 as the first member of a new class of anti-HIV drugs-HIV fusion inhibitors.
HIV fusion with the host cell is initiated by binding of Env surface subunit 120 to the primary receptor CD4 and a co-receptor, CXCR4 or CCR5, resulting in a series of conformational changes in gp41, including insertion of FP into the target cell membrane and association of CHR-helices with the NHR-trimer, a prehairpin intermediate, to form a stable six-helix bundle (6-HB) core, which bring the viral envelope and target cell membrane into close proximity for fusion. X-ray crystallographic studies have shown that 6-HB consists of three molecules of a NHR-peptide that form the inner trimeric coiled-coil and three copies of a CHR-peptide that pack obliquely in an anti-parallel configuration into the highly conserved hydrophobic grooves on the surface of the internal NHR-trimer. Each groove has a deep hydrophobic pocket (FIG. 1B, circled at bottom), which plays an important role in viral fusion and maintaining the stability of the 6-HB. Accordingly, it is proposed that a CHR-peptide (e.g., enfuvirtide or C34) inhibits HIV-1 fusion by binding to the viral gp41 NHR-trimer at the fusion-intermediate state to block the formation of fusion-active core of gp41. Therefore, the gp41 NHR-trimer is a crucial target for HIV therapeutics, and may also serve as an important target for HIV vaccines.
However, the gp41 NHR-peptides cannot form stable and soluble trimers in vitro spontaneously because these peptides have tendency to aggregate in physiological solutions. To study the structure, function and immunogenicity of the gp41 prehairpin intermediate, several soluble and stable NHR-trimer mimetics have been created, including IQN17 (FIG. 1C), in which a 17-mer NHR-peptide (aa 565-581) involving in formation of the gp41 hydrophobic pocket is linked with GCN4-pIQI (IQ) motif, a soluble trimeric coiled coil, and similar mimetics with higher stability, including IZN17, IZN36 and (ccIZN17)3, in which IQ is replaced with more stable trimerization motif, IZ. Other examples of NHR-trimer mimetics include NCCG-gp41, N35CCG-N13, and 5-Helix in which one CHR peptide of the 6-HB is missing so as to expose the groove of the NHR-trimer. Although all these NHR-trimer mimetics properly present the hydrophobic groove and pocket and are effective in interacting with viral gp41 CHR to inhibit HIV-1 fusion, none of them could induce detectable neutralizing antibody responses in immunized animals. It is believed that the accessibility of the prehairpin intermediate of gp41 to antibody molecules (e.g., IgG) is limited because antisera directed against NHR-peptides exhibited no neutralizing activity at 37° C., but were effective under suboptimal temperature (31.5° C.) to prolong fusion intermediates. But interestingly, IgG1 m44, a human mAb directed against gp41 is much more potent than Fab m44 in neutralizing infection by primary HIV-1 isolates. Another human mAb, D5, that specifically binds to the pocket of NHR-trimer is highly potent to neutralize HIV-1 infection. Although rabbit antisera induced against N35CCG-N13 showed no neutralizing activity, purified IgG from the antisera (about 5-10% of total IgGs) with high-binding affinity to the NHR-trimer could significantly inhibit HIV-1 Env-mediated cell fusion. These data suggest that the gp41 NHR-trimer in the prehairpin fusion intermediate state is accessible to antibodies, which is not restricted by either antibody size or the presence of a kinetic barrier, but may be limited by the affinity of antibodies to bind with the NHR-trimer. Therefore, it is essential to design an immunogen with proper conformation and increased immunogenicity that can induce antibodies with high-binding affinity to the gp41 prehairpin intermediate.